PHOTOAFFINITY-LABELED LIGAND-BINDING DOMAINS ON DOPAMINE TRANSPORTERSIDENTIFIED BY PEPTIDE-MAPPING

Binding domains on rat dopamine transporters for cocaine and diphenylm ethoxy)ethyl-4-(3-phenylpropyl)piperazine compounds were identified us ing controlled proteolysis of photoaffinity-labeled protein and epitop e-specific immunoprecipitation of the labeled fragments. Rat dopamine transporters were photoaffinity labeled with [2-(diphenylmethoxy)ethyl ]-4-[2-(4-azido-3-[I-125] iodophenyl)ethyl]piperazine ([I-125]DEEP) di phenylmethoxy)ethyl-4-(3-phenylpropyl)piperazine analog] or 3 beta-(rh o-chlorophenyl)tropane-2 beta-carboxylic acid, 4'-azido-3'-[I-125] iod ophenylethyl ester ([I-125]RTI 82) (a cocaine analog) and were gel pur ified to remove contaminating radioactivity. The resulting samples wer e treated with V8 protease or trypsin and analyzed by sodium dodecyl s ulfate-polyacrylamide gel electrophoresis. The peptide maps generated with each enzyme were different for each of the ligands, suggesting th at the ligands were incorporated into different regions of the protein . Identical peptide maps were generated from striatum- and nucleus acc umbens-derived transporters, indicating that these polypeptides are hi ghly similar in primary sequence. The proteolytic fragments generated by V8 protease were localized to specific domains of the protein using antipeptide antibodies corresponding to five different regions of the transporter. Fragments of 10 and 7 kDa from [I-125]DEEP-labeled trans porters were specifically immunoprecipitated with an antibody generate d against amino acids 42-59 (near the first putative transmembrane dom ain), whereas a 34-kDa fragment from [I-125]RTI 82-labeled transporter s was precipitated with three different sera corresponding to regions in the carboxyl-terminal two thirds of the protein. None of the V8 fra gments smaller than 45 kDa, containing either photolabel, was altered in molecular mass by N-deglycosylation. The results indicate that phot oincorporation of [I-125]DEEP occurs in the amino half of the dopamine transporter, near the first two transmembrane helices, whereas [I-125 ]RTI 82 labels the carboxyl-terminal region of the protein, between tr ansmembrane domains 4 and 12.